The overall objectives of this project are to begin studies to understand the vitamin K cycle, which is important for blood coagulation, bone formation, and probably other physiological processes. The project is divided into four specific aims; for the first three we will continue our work on the structure and function of the vitamin K-dependent enzyme gammaglutamyl carboxylase. We will use alanine-scanning mutagenesis for the first two aims: to determine the propeptide and vitamin K binding sites on the carboxylase molecule and to characterize a naturally-occurring carboxylase gene mutation found in a patient. Since the structure of the carboxylase molecule is essential to understanding its function our third aim will be to analyze both topology and 3-dimensional structure. The method of topological analysis we have chosen is particularly well-suited to integral membrane proteins such as the carboxylase, as is our method of electron diffraction to determine 3-dimensional structure. Finally, for our fourth specific aim, we will focus on another enzyme important in the vitamin K cycle, the vitamin K epoxide reductase. We will begin work to purify and characterize this protein, about which little is currently known. One of our long-term goals is to combine the carboxylase and vitamin K epoxide reductase, along with any other essential components, in an in vitro system to start to reconstitute the entire vitamin K cycle. These studies have significant clinical relevance in that they could lead to improved therapies for blood coagulation and thrombosis disorders.